Encapsulation of Nanoparticles with Statistical Copolymers with Different Surface Charges and Analysis of Their Interactions with Proteins and Cells.

Encapsulation with polymers is a well-known strategy to stabilize and functionalize nanomaterials and tune their physicochemical properties. Amphiphilic copolymers are promising in this context, but their structural diversity and complexity also make understanding and predicting their behavior challenging. This is particularly the case in complex media which are relevant for intended applications in medicine and nanobiotechnology. Here, we studied the encapsulation of gold nanoparticles and quantum dots with amphiphilic copolymers differing in their charge and molecular structure. Protein adsorption to the nanoconjugates was studied with fluorescence correlation spectroscopy, and their surface activity was studied with dynamic interfacial tensiometry. Encapsulation of the nanoparticles without affecting their characteristic properties was possible with all tested polymers and provided good stabilization. However, the interaction with proteins and cells significantly depended on structural details. We identified statistical copolymers providing strongly reduced protein adsorption and low unspecific cellular uptake. Interestingly, different zwitterionic amphiphilic copolymers showed substantial differences in their resulting bio-repulsive properties. Among the polymers tested herein, statistical copolymers with sulfobetaine and phosphatidylcholine sidechains performed better than copolymers with carboxylic acid- and dimethylamino-terminated sidechains.

Structural Analysis and Intrinsic Enzyme Mimicking Activities of Ligand-Free PtAg Nanoalloys.

Nanozymes are nanomaterials with biocatalytic properties under physiological conditions and are one class of artificial enzymes to overcome the high cost and low stability of natural enzymes. However, surface ligands on nanomaterials will decrease the catalytic activity of the nanozymes by blocking the active sites. To address this limitation, ligand-free PtAg nanoclusters (NCs) are synthesized and applied as nanozymes for various enzyme-mimicking reactions. By taking advantage of the mutual interaction of zeolitic imidazolate frameworks (ZIF-8) and Pt precursors, a good dispersion of PtAg bimetal NCs with a diameter of 1.78 ± 0.1 nm is achieved with ZIF-8 as a template. The incorporation of PtAgNCs in the voids of ZIF-8 is confirmed with structural analysis using the atomic pair-distribution function and powder X-ray diffraction. Importantly, the PtAgNCs present good catalytic activity for various enzyme-mimicking reactions, including peroxidase-/catalase- and oxidase-like reactions. Further, this work compares the catalytic activity between PtAg NCs and PtAg nanoparticles with different compositions and finds that these two nanozymes present a converse dependency of Ag-loading on their activity. This study contributes to the field of nanozymes and presents a potential option to prepare ligand-free bimetal biocatalysts with sizes in the nanocluster regime.

Quantitative Assessment of Endosomal Escape of Various Endocytosed Polymer-Encapsulated Molecular Cargos upon Photothermal Heating.

Encapsulated molecular cargos are efficiently endocytosed by cells. For cytosolic delivery, understanding the dynamic process of cargos release from the carrier vehicles used for encapsulation and the lysosomes where the carrier vehicles are trapped (which in general is the bottleneck), followed by diffusion in the cytosol is important for improving drug/gene delivery strategies. A methodology is reported to image this process on a millisecond scale and to quantitatively analyze the data. Polyelectrolyte capsules with embedded gold nanostars to encapsulate 43 fluorescent molecular cargos with diverse properties, ranging from small fluorophores to fluorescently labeled proteins, siRNA, etc., are used. By short laser irradiation intracellular release of the molecular cargos from endocytosed capsules into the cytosol is triggered, and their intracellular spreading is imaged. Most of the released molecular cargos evenly distribute inside the entire cell, while others are enriched in certain cell compartments. The time the different molecular cargos take to distribute within cells, i.e., the spreading time, is used as a quantifier. Quantitative analysis reveals that intracellular spread cannot be described by free diffusion, but is determined by interaction of the molecular cargo with intracellular components.

Assembly and Degradation of Inorganic Nanoparticles in Biological Environments.

In solution, nanoparticles may be conceptually compartmentalized into cores and engineered surface coatings. Recent advances allow for simple and accurate characterization of nanoparticle cores and surface shells. After introduction into a complex biological environment, adsorption of biological molecules to the nanoparticle surface as well as a loss of original surface components occur. Thus, colloidal nanoparticles in the context of the biological environment are hybrid materials with complex structure, which may result in different chemical, physical, and biological outcomes as compared to the original engineered nanoparticles. In this review, we will discuss building up an engineered inorganic nanoparticle from its inside core to its outside surface and following its degradation in a biological environment from its outside to its inside. This will involve the way to synthesize selected inorganic nanoparticles. Then, we will discuss the environmental changes upon exposure of these nanoparticles to biological media and their uptake by cells. Next, the intracellular fate of nanoparticles and their degradation will be discussed. Based on these examples, the need to see nanoparticles in the context of the biological environment as dynamic hybrid materials will be highlighted.

Laterally and Temporally Controlled Intracellular Staining by Light-Triggered Release of Encapsulated Fluorescent Markers.

Fluorescent molecular markers were encapsulated. The capsules were additionally modified with plasmonic nanoparticles. The encapsulated markers were endocytosed by cells. Upon light stimulation the plasmonic nanoparticles generated heat, which opened the encapsulation and transiently perforated the endosomal/lysosomal membrane surrounding the capsule, thus allowing for release of the marker into the cytosol. Fluorescence labeling of different intracellular compartments was demonstrated in this way. Most important, the cells do not need to be fixed and perforated, as the molecular markers are introduced into cells by endocytosis and subsequent light-induced release. Thus this technique allows for intracellular fluorescence labeling of living cells.

Polymer-coated nanoparticles: Carrier platforms for hydrophobic water- and air-sensitive metallo-organic compounds.

Many of the relevant compounds for anticancer therapy are metal-based compounds (metallodrugs), being platinum-based drugs such as cisplatin, carboplatin (Paraplatin®), and oxaliplatin (Eloxatin®) the most widely used. Despite this, their application is limited by issues such as cell-acquired platinum resistance and manifold side effects following systemic delivery. Thus, the development of new metal-based compounds is highly needed. The catalytic properties of a variety of metal-based compounds are nowadays very well known, which opens new opportunities to take advantage of them inside living cells or organisms. However, many of these compounds are hydrophobic and thus not soluble in aqueous solution, as they lack stability against water or oxygen presence. Thus, versatile platforms capable of enhancing the features of these compounds in aqueous solutions are of importance in the development of new drugs. Surface engineered nanoparticles may render metallodrugs with good colloidal stability in water and in complex media containing high salt concentration and/or proteins. Herein, polymer coated nanoparticles are proposed as a platform to link insoluble and water/oxygen sensitive drugs. The linkage of insoluble and oxygen sensitive tin clusters to nanoparticles is presented, aiming to enhance both, the solubility and the stability of these compounds in water, which may be an alternative approach in the development of metal-based drugs. The formation of the cluster-nanoparticle system was confirmed via inductively coupled plasma mass spectrometry experiments. The catalytic activity and the stability of the cluster in water were studied through the reduction of methylene blue. Results demonstrate that in fact the tin clusters could be transferred into aqueous solution and retained their catalytic activity.

Influence of Temperature on the Colloidal Stability of Polymer-Coated Gold Nanoparticles in Cell Culture Media.

The temperature-dependence of the hydrodynamic diameter and colloidal stability of gold-polymer core-shell particles with temperature-sensitive (poly(N-isopropylacrylamide)) and temperature-insensitive shells (polyallylaminine hydrochloride/polystyrensulfonate, poly(isobutylene-alt-maleic anhydride)-graft-dodecyl) are investigated in various aqueous media. The data demonstrate that for all nanoparticle agglomeration, i.e., increase in effective nanoparticle size, the presence of salts or proteins in the dispersion media has to be taken into account. Poly(N-isopropylacrylamide) coated nanoparticles show a reversible temperature-dependent increase in size above the volume phase transition of the polymer shell when they are dispersed in phosphate buffered saline or in media containing protein. In contrast, the nanoparticles coated with temperature-insensitive polymers show a time-dependent increase in size in phosphate buffered saline or in medium containing protein. This is due to time-dependent agglomeration, which is particularly strong in phosphate buffered saline, and induces a time-dependent, irreversible increase in the hydrodynamic diameter of the nanoparticles. This demonstrates that one has to distinguish between temperature- and time-induced agglomerations. Since the size of nanoparticles regulates their uptake by cells, temperature-dependent uptake of thermosensitive and non-thermosensitive nanoparticles by cells lines is compared. No temperature-specific difference between both types of nanoparticles could be observed.

Basic Physicochemical Properties of Polyethylene Glycol Coated Gold Nanoparticles that Determine Their Interaction with Cells.

A homologous nanoparticle library was synthesized in which gold nanoparticles were coated with polyethylene glycol, whereby the diameter of the gold cores, as well as the thickness of the shell of polyethylene glycol, was varied. Basic physicochemical parameters of this two-dimensional nanoparticle library, such as size, ζ-potential, hydrophilicity, elasticity, and catalytic activity ,were determined. Cell uptake of selected nanoparticles with equal size yet varying thickness of the polymer shell and their effect on basic structural and functional cell parameters was determined. Data indicates that thinner, more hydrophilic coatings, combined with the partial functionalization with quaternary ammonium cations, result in a more efficient uptake, which relates to significant effects on structural and functional cell parameters.

Particle-based optical sensing of intracellular ions at the example of calcium - what are the experimental pitfalls?

Colloidal particles with fluorescence read-out are commonly used as sensors for the quantitative determination of ions. Calcium, for example, is a biologically highly relevant ion in signaling, and thus knowledge of its spatio-temporal distribution inside cells would offer important experimental data. However, the use of particle-based intracellular sensors for ion detection is not straightforward. Important associated problems involve delivery and intracellular location of particle-based fluorophores, crosstalk of the fluorescence read-out with pH, and spectral overlap of the emission spectra of different fluorophores. These potential problems are outlined and discussed here with selected experimental examples. Potential solutions are discussed and form a guideline for particle-based intracellular imaging of ions.

Ion transport through polyelectrolyte multilayers.

Polyelectrolyte multilayer (PEM) films and capsules loaded with ion-sensitive fluorophores can be used as ion-sensors for many applications including measurements of intracellular ion concentration. Previous studies have shown the influence of the PEM films/shells on the specific response of encapsulated ion-sensitive fluorophores. PEM shells are considered as semipermeable barriers between the environment and the encapsulated fluorophores. Parameters such as the time response of the encapsulated sensor can be affected by the porosity and charge of the PEM shell. In this study, the time response of an encapsulated pH-sensitive fluorophore towards pH changes in the surrounding environment is investigated. Furthermore, the conductance of PEM films for potassium ions is determined.

Integration of organic fluorophores in the surface of polymer-coated colloidal nanoparticles for sensing the local polarity of the environment.

Inorganic hydrophobically capped NPs such as quantum dots, superparamagnetic iron oxide, or gold nanoparticles can be modified to make them water-soluble by their embedding in an amphiphilic polymer shell. This polymer shell can be prefunctionalized by the integration of organic fluorophores, which allows the observation of the nanoparticles with fluorescence based techniques. The fluorophore could be either located more in the hydrophobic part of the inner polymer shell, or on the hydrophilic surface pointing towards solution. Herein we prepared gold nanoparticles decorated with the organic fluorophore FE, 4'-N,N-diethylamino-3-hydroxyflavone (FE), which possesses fluorescence sensitive to the polarity and hydrogen-bonding properties of the surrounding local environment. Based on the response of FE in the polymer shell to isopropanol, and CTAB compared to the response of free FE we conclude that the FE fluorophore is situated within the inner polymer shell. Nevertheless the fluorophore in the polymer shell can still sense polarity changes in solution.

Quantification of the internalization patterns of superparamagnetic iron oxide nanoparticles with opposite charge.

Time-resolved quantitative colocalization analysis is a method based on confocal fluorescence microscopy allowing for a sophisticated characterization of nanomaterials with respect to their intracellular trafficking. This technique was applied to relate the internalization patterns of nanoparticles i.e. superparamagnetic iron oxide nanoparticles with distinct physicochemical characteristics with their uptake mechanism, rate and intracellular fate.The physicochemical characterization of the nanoparticles showed particles of approximately the same size and shape as well as similar magnetic properties, only differing in charge due to different surface coatings. Incubation of the cells with both nanoparticles resulted in strong differences in the internalization rate and in the intracellular localization depending on the charge. Quantitative and qualitative analysis of nanoparticles-organelle colocalization experiments revealed that positively charged particles were found to enter the cells faster using different endocytotic pathways than their negative counterparts. Nevertheless, both nanoparticles species were finally enriched inside lysosomal structures and their efficiency in agarose phantom relaxometry experiments was very similar.This quantitative analysis demonstrates that charge is a key factor influencing the nanoparticle-cell interactions, specially their intracellular accumulation. Despite differences in their physicochemical properties and intracellular distribution, the efficiencies of both nanoparticles as MRI agents were not significantly different.

Biodegradable particles for protein delivery: Estimation of the release kinetics inside cells.

A methodology to quantify the efficiency of the protein loading and in-vitro delivery for biodegradable capsules with different architectures based on polyelectrolytes (dextran sulfate, poly-L-arginine and polyethylenimine) and SiO2 was developed. The capsules were loaded with model proteins such as ovalbumin and green fluorescent protein (GFP), and the protein release profile inside cells (either macrophages or HeLa cells) after endocytosis was analysed. Both, protein loading and release kinetics were evaluated by analysing confocal laser scanning microscopy images using MatLab and CellProfiler software. Our results indicate that silica capsules showed the most efficient release of proteins as cargo molecules within 48 h, as compared to their polymeric counterparts. This developed method for the analysis of the intracellular cargo release kinetics from carrier structures could be used in the future for a better control of drug release profiles.

Synthesis and characterization of ratiometric ion-sensitive polyelectrolyte capsules.

Micrometer-sized polyelectrolyte capsules are synthesized, which have ion-sensitive fluorophores embedded in their cavities. As the membranes of the capsules are permeable to ions, the fluorescence of the capsules changed with the ion concentration. In particular, capsules sensitive to protons, sodium, potassium, and chloride ions are fabricated and their fluorescence response analyzed. In order to allow for ratiometric measurements, additional fluorophores whose emission do not depend on the ion concentration and which emit a different wavelength are co-embedded in the capsule cavities.

Polymer-coated nanoparticles: a universal tool for biolabelling experiments.

Water solubilization of nanoparticles is a fundamental prerequisite for many biological applications. To date, no single method has emerged as ideal, and several different approaches have been successfully utilized. These 'phase-transfer' strategies are reviewed, indicating key advantages and disadvantages, and a discussion of conjugation strategies is presented. Coating of hydrophobic nanoparticles with amphiphilic polymers provides a generic pathway for the phase transfer of semiconductor, magnetic, metallic, and upconverting nanoparticles from nonpolar to polar environments. Amphiphilic polymers that include maleimide groups can be readily functionalized with chemical groups for specific applications. In the second, experimental part, some of the new chemical features of such polymer-capped nanoparticles are demonstrated. In particular, nanoparticles to which a pH sensitive fluorophore has been attached are described, and their use for intracellular pH-sensing demonstrated. It is shown that the properties of analyte-sensitive fluorophores can be tuned by using interactions with the underlying nanoparticles.

Gene silencing mediated by magnetic lipospheres tagged with small interfering RNA.

Lipospheres made from soy bean oil and a combination of the cationic lipid Metafectene and the helper lipid dioleoylphosphatidyl-ethanolamine were functionalized with magnetic nanoparticles (NPs) and small interfering RNA (siRNA). The resulting magnetic lipospheres loaded with siRNA are proven here as efficient nonviral vectors for gene silencing. Embedding magnetic NPs in the shell of lipospheres allows for magnetic force-assisted transfection (magnetofection) as well as magnetic targeting in both static and fluidic conditions mimicking the bloodstream.

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle.

The protein corona can significantly modulate the physicochemical properties and gene delivery of polyethylenimine (PEI)/DNA complexes (polyplexes). The effects of the protein corona on the transfection have been well studied in terms of averaged gene expression in a whole cell population. Such evaluation methods give excellent and reliable statistics, but they in general provide the final transfection efficiency without reflecting the dynamic process of gene expression. In this regard the influence of bovine serum albumin (BSA) on the gene expression of PEI polyplexes also on a single cell level via live imaging is analyzed. The results reveal that although the BSA corona causes difference in the overall gene expression and mRNA transcription, the gene expression behavior on the level of individual cell is similar, including the mitosis-dependent expression, distributions of onset time, expression pattern in two daughter cells, and expression kinetics in successfully transfected cells. Comparison of single cell and ensemble data on whole cell cultures indicate that the protein corona does not alter the transfection process after nuclear entry, including cell division, polyplex dissociation, and protein expression. Its influence on other steps of in vitro gene delivery before nuclear entry shall render the difference in the overall transfection.

Hyperspectral-enhanced dark field analysis of individual and collective photo-responsive gold-copper sulfide nanoparticles.

We used hyperspectral-enhanced dark field microscopy for studying physicochemical changes in biomaterials by tracking their unique spectral signatures along their pathway through different biological environments typically found in any biomedical application. We correlate these spectral signatures with discrete environmental features causing changes in nanoparticles' physicochemical properties. We use this correlation to track the nanoparticles intracellularly and to assess the impact of these changes on their functionality. We focus on one example of a photothermal nanocomposite, i.e., polymer-coated gold/copper sulfide nanoparticles, because their performance depends on their localized surface plasmon peak, which is highly sensitive to environmental changes. We found spectral differences both in the dependence of time and discrete environmental factors, affecting the range of illumination wavelengths that can be used to activate the functionality of these types of nanoparticles. The presence of proteins (protein corona) and the increase in ionic strength induce a spectral broadening towards the NIR region which we associated with nanoparticles' agglomeration. In acidic environments, such as that of the lysosome, a red shift was also observed in addition to a decrease in the scattering intensity probably associated with a destabilization of the proteins and/or the change in the net charge of the polymer around the nanoparticles. We observed a loss of the photo-excitation potential of those nanoparticles exposed to acidic conditions in the <600 nm spectral rage. In a similar manner, ageing induces a transitioning from a broad multipeak spectrum to a distinct shoulder with time (up to 8 months) with the loss of spectral contribution in the 450-600 nm range. Hence, a fresh preparation of nanoparticles before their application would be recommended for an optimal performance. We highlight the impact of ageing and the acidic environment on the responsiveness of this type of plasmonic nanoparticle. Regardless of the spectral differences found, polymer-coated gold/copper sulfide nanoparticles retained their photothermal response as demonstrated in vitro upon two-photon irradiation. This could be ascribed to their robust geometry provided by the polymer coating. These results should be useful to rationally design plasmonic photothermal probes.

Influence of the chirality of carbon nanodots on their interaction with proteins and cells.

Carbon nanodots with opposite chirality possess the same major physicochemical properties such as optical features, hydrodynamic diameter, and colloidal stability. Here, a detailed analysis about the comparison of the concentration of both carbon nanodots is carried out, putting a threshold to when differences in biological behavior may be related to chirality and may exclude effects based merely on differences in exposure concentrations due to uncertainties in concentration determination. The present study approaches this comparative analysis evaluating two basic biological phenomena, the protein adsorption and cell internalization. We find how a meticulous concentration error estimation enables the evaluation of the differences in biological effects related to chirality.

Liposome-based measurement of light-driven chloride transport kinetics of halorhodopsin.

We report a simple and direct fluorimetric vesicle-based method for measuring the transport rate of the light-driven ions pumps as specifically applied to the chloride pump, halorhodopsin, from Natronomonas pharaonis (pHR). Previous measurements were cell-based and methods to determine average single channel permeability challenging. We used a water-in-oil emulsion method for directional pHR reconstitution into two different types of vesicles: lipid vesicles and asymmetric lipid-block copolymer vesicles. We then used stopped-flow experiments combined with fluorescence correlation spectroscopy to determine per protein Cl- transport rates. We obtained a Cl- transport rate of 442 (±17.7) Cl-/protein/s in egg phosphatidyl choline (PC) lipid vesicles and 413 (±26) Cl-/protein/s in hybrid block copolymer/lipid (BCP/PC) vesicles with polybutadine-polyethylene oxide (PB12PEO8) on the outer leaflet and PC in the inner leaflet at a photon flux of 1450 photons/protein/s. Normalizing to a per photon basis, this corresponds to 0.30 (±0.07) Cl-/photon and 0.28 (±0.04) Cl-/photon for pure PC and BCP/PC hybrid vesicles respectively, both of which are in agreement with recently reported turnover of ~500 Cl-/protein/s from flash photolysis experiments and with voltage-clamp measurements of 0.35 (±0.16) Cl-/photon in pHR-expressing oocytes as well as with a pHR quantum efficiency of ~30%.